Induced cell motility functions during organogenesis and wound repair to (re)populate the target space, a situation that is deficient in a number of clinical conditions and in aging individuals. During tissue (re)generation stromal fibroblasts, endothelial cells, and possibly circulating stem cells migrate into the provisional matrix from surrounding tissues to form both the supporting matrix and vasculature. The initial cell immigration is directional, being driven by signals, 'cues' that arise from within the wound bed. However, once within the wound bed, the cells must distribute without evident stimuli gradients. Thus, our long-term goal is to determine how cells establish and then maintain progressive motility to repopulate tissues in response to external stimuli. Cell motility requires asymmetry of biophysical cell processes, which in eukaryotic cells is established by intracellular signals, in response to cytokine 'cues' in the environment, mainly via the EGF receptor for stromal and stems cells and VEGF for endothelial cells, though signaling via other receptors also follows the same intracellular cascades. Cells must extend lamellipodia and stabilize the dominant protrusion, while rear de-adhesion and retraction is required to enable progressive movement. Between these two cell regions, contractility occurs to bring the cell body forward. Overriding these processes, a cell must establish its directionality, intrinsically in absence of significant external gradients. During the initial two grant periods it was found that a key is the asymmetric distribution of PI(4,5)P2 (phosphoinositide 4,5 bisphosphate; for ease of reading we will not be using the subscript form) and its processing. Interestingly, it is becoming increasingly evident that PIP2 functions not only in its known role as a docking site and precursor to active metabolites but in the novel role of active signal transducer and cofactor in and of itself. Recent work suggests that PIP2 directly activates calpain 2 to actuate rear release, may serve to direct the transcellular contractility needed to move the cell body forward, and may regulate actin cytoskeleton bundling. Thus, our findings allow advance beyond our original hypothesis that intracellular signaling cascades were subcellularly localized to positing a unifying mechanism for asymmetry during growth factor-induced motility. We hypothesize that the localized activation of key biochemical signaling cascades required for productive cell motility results from the asymmetric actions of phospho-inositide, by both docking/localizing and directly modulating effector proteins. Our Specific Aims propose to test the following postulates: I. That m-calpain (calpain-2) is directly activated by PIP2. II. That 1-actinin-4 (ACTN4) regulation by phospho-inositides controls the linkage between the cytoskeleton and the membrane. III. That distribution of phospho-inositides and phospho-inositide turnover direct contractility. The completion of these investigations will define molecular bases of the spatial restriction of receptor signaling pathways and resultant biophysical responses critical to human tissue cell migration, providing missing information for basic biology of cell functioning. This knowledge will enable the design on a subcellular scale of 'smart' scaffolds for cell and tissue engineering directing the synthesis of both the matrix and the vascular bed that is required to support tissue function. PUBLIC HEALTH RELEVANCE: The orchestrated movement of many types of cells, controlled by signals from the external milieu or micro-environment, is crucial for organogenesis and wound repair. An outstanding question remains how a cell determines how to organize its intracellular machinery for productive motility. Testing the proposed foundational model of phospho-inositide distribution localizing the biochemical and biophysical processes would provide novel insights into the basic cell biology of wound repair and allow for future development of smart surfaces that could harness this information to direct reparative cells into regions of injury.